Mitochondrial biogenesis and the cytoplasmic genetics of mammalian cells in culture will be investigated through mutational analysis and use of techniques in cell biology, biochemistry and somatic cell genetics. The principal projects to be undertaken are: a) The genetic properties of cytoplasmically-inherited PYR-IND mutants (pyruvate-independent expression of chloramphenicol resistance) will be analyzed. The actual PYR-IND mutation frequency will be determined by fluctuation analysis; the preliminary indications of marked differences in frequency among cell lines will be extended; and the role of the mtDNA in determining the mutation frequency tested. b) Mutations within the mtDNA will be sought by selection for resistance to known inhibitors of various mitochondrial functions--electron transport, oxidative phosphorylation, protein synthesis. A procedure for selective cytoplasmic gene mutagenesis is proposed. c) Formal genetic analysis of mitochondrial DNA will be undertaken using the CAP-R and PYR-IND markers and any new cytoplasmic mutations isolated. Multi-factorial hybrid crosses will be used to analyze transmission, reassortment and recombination of mitochondrial gene loci. d) The biochemical basis of the PYR-IND mutant phenotype and its effects on various metabolic pathways will be studied; one possibility to be investigated is that this phenotype is due to an impairment in mitochondrial respiration or oxidative phosphorylation. The biochemical properties of other new mitochondrial mutations isolated in this laboratory will be investigated as well: mitochondrial respiration, oligomycin-sensitive ATPase, oxidative phosphorylation capacity and mitochondrial protein synthesis. e) The loci analyzed genetically will then be physically mapped within the mtDNA using standard restriction enzyme procedures.